Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace–1) being a new locus and the other (ace–2) being homologous to the gene previously described in Drosophila. The gene ace–1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace–1 and ace–2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace–1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace–1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace–2 in at least the Drosophilidae and Muscidae and ace–1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.